Molecular Basis of Antibody Diversity

-Dr. Nilesh Chandra

Objectives

• Antibody: structure and properties

• Genetic organisation

• Genetic rearrangement

• Antibody diversity

Antibody

• Glycoprotein.• Produced by plasma cells.• Recognize & bind antigens.• Lead to:– Phagocytosis– Complement activation– Antibody dependent cell cytotoxicity (ADCC)

Antibody- structure

Functions of Antibodies

• B cell Ag receptor as mAb.• Neutralization of Ag by sAb.• Complement activation• Opsonization• ADCC• Mucosal immunity• Neonatal immunity (by IgG & IgA)• Immediate hypersensitivity by IgE

Immunoglobulin gene families

• Located on different chromosomes for different chains:– H-chain gene family on Ch 14.– Kappa light chain gene family on Ch 2.– Lambda light chain gene family on Ch 22.

• One family multiple gene segments.• One gene segment multiple genes for

same specific region.

Light Chain genes

• Encoded by 3 genes:– V (variable gene)– J (joining gene)– C (constant gene)

• V & J together code for variable region ( VL).

• C gene codes for the constant region ( CL).• A complete L chain formed by splicing of V, J &

C genes.

Light Chain genes

Heavy Chain genes

• Encoded by 4 genes:– Variable region coded by 3 genes:• VH (variable)

• JH (joining)

• DH (diversity)

–Constant region (CH) encoded by one single gene.–H-chain C-segment has 9 genes.

Heavy Chain genes

Gene rearrangement

• At DNA level• At RNA level

Rearrangement at DNA level

• V,D & J joining in H-chains; V & J joining in L-chains.

• C region genes remain separated from V-region gene by J genes and intron.

• A primary RNA transcript is generated.• Only Cμ & Cδ genes are transcribed in naïve B

cells.• The other C region genes transcribed during

class switching.

Gene rearrangement

• At DNA level• At RNA level

Rearrangement at RNA level

• Takes place during the primary RNA transcript processing.

• The C-region gene spliced with V-region genes to generate complete H & L chain genes.

• Plays crucial role in:– Production of membrane bound Ab.– Production of secreted form of Ab.– Production of different classes of Ab.

Heavy chain gene rearrangement

• Rearranged V region at DNA level conatins the following sequences from 5’ end to 3’ end:– L-exon of joined VH gene.

– VHDHJH –combined exon.

– Intron between L & VHDHJH.– Intron 3’ to J gene.– Remaining JH genes, if any, followed by complete

set of CH region genes.

Heavy chain gene rearrangement

• The rearranged H-gene is transcribed.• Next rearrangement at RNA level takes place.• Differential splicing of the primary RNA

transcript leads to generation of mRNA for both μ and δ heavy chains.

• After translational, leader peptide is cleaved to produce a fully functional chain.

Light chain gene rearrangement

• Occurs after the rearrangement in heavy chain.

• Similar in pattern.• No D-genes involved.– Variable region rearrangement in DNA.– Splicing with C-region gene during RNA

processing.

Antibody Diversity Mechanism

• Multigene organisations of Ig chain.• Combinatorial joining of variable region genes.• Junctional flexibility.• P-nucleotide addition.• N-nucleotide addition.• Somatic hypermutation.• Combinatorial association of H- and L-chain.

Multigene organisations of Ig chain

• Large number of variable region genes:

• David Parry

Gene segment

Number of genes

Heavy chain Kappa chain Lambda chain

V 51 40 30

D 27 0 0

J 6 5 4

Combinatorial joining of variable region genes

• Random rearrangement of genes from various segments.

• Vast variety of combinations are possible due to this random joining.

Because of the diversity contributed by junctional flexibility, P- & N-region nucleotide addition, and somatic mutation, the actual potential exceeds these estimates by several orders of magnitude.

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Junctional flexibility

P-nucleotide addition

• During DNA rearrangement, a hair-pin structure is formed.• This hairpin is later cleaved by an endonuclease.• A short single strand at the end of the coding sequence is

left.• The subsequent addition of complementary nucleotides

to this strand (P-addition) by repair enzymes generates a palindromic sequence in the coding joint, hence called P-nucleotides.

• Variation in the position at which the hairpin is cut thus leads to variation in the sequence of the coding joint.

N-nucleotide addition

• Addition of new nucleotides at the free 3’ end.• By enzyme terminal deoxynucleotide

transferase (TdT).• Up to 15 nucleotides can be added at the

coding joints.• Contribute to antibody diversity.

Somatic hypermutation

• V-region genes in B-cell, on antigenic stimulation, undergo point mutations.

• Mutations result from nucleotide substitution.• Seen in response to T-cell dependent protein

antigens.• Influence the process of affinity maturation.

Combinatorial association of H- and L-chain

• Specificity of antibody is determined by the V-region of both the H & L chains.

• The possible combinations of the H and L chains are also a source of antibody diversity.

Because of the diversity contributed by junctional flexibility, P- & N-region nucleotide addition, and somatic mutation, the actual potential exceeds these estimates by several orders of magnitude.

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Multiple myeloma

• Malignant disease of plasma cells.• Normally, H & L chains are produced in equal

amounts.• In multiple myeloma, L-chains are synthesized

much in excess than H-chains.• Urine of these patients show increased

amounts of L-chain secretion.

Clinical applications

• Understanding of immunoglobulin structure and formation has opened up a new world of possibilities:– Monoclonal antibodies– Engineering mice with human immune systems– Generating chimeric and hybrid antibodies for

clinical use– Abzymes: antibodies with enzyme capability

Summary

• Antibodies and their functions.

• Immunoglobulin gene families (H & L).

• Gene rearrangement (H & L).

• Mechanisms of Antibody diversity.

• Clinical applications.

References

• Immunology, 5th Edition; Kuby• Roitt’s Essential Immunology, 10th edition• Essentials of Immunology; S.K.Gupta• Diverse functions for DNA and RNA editing in the

immune system. Hamilton CE, Papavasiliou FN, Rosenberg BR. RNA Biol. 2010 Mar-Apr;7(2):220-8. Epub 2010 Mar 29.

• B cells from the bench to the clinical practice. Moura R, Agua-Doce A, Weinmann P, Graça L, Fonseca JE. Acta Reumatol Port. 2008 Apr-Jun;33(2):137-54.

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